talk_Cav

A PHYSICAL MODEL FORCAVEOLAR MEMBRANES : 

A PHYSICAL MODEL FORCAVEOLAR MEMBRANES Matthew Turner & Alun Evans (Univ. Warwick - U.K) Pierre Sens (CNRS: Inst. Charles Sadron - Strasbourg & Inst. Curie - Paris ) http://perso.curie.fr/Pierre.Sens/ Pierre.Sens@curie.fr

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hydrophobic hydrophilic Membranes Proteins :

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“Curvature active” Proteins : Drive Membrane Deformation Endocytosis - Cell fusion Membrane recycling … Concentrate binding sites Cell signalling … Ex: endocytosis by formation of Clathrin coated pits Ex. Caveolae receptors Target molecules

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Physical model for fluid membranes Equilibrium properties Balance between hydrophobic attraction steric, electrostatic repulsion Bending the membrane cost energy fairly small C = curvature Bending modulus The membrane in under tension Energy Energy = area increase

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Example - Thermal fluctuations of membranes Monge representation Curvature Deformation energy With surface tension Curvature dominates the small lengthscales From R. Dimova mpikg-golm 50nm < l < Large biological ”free membranes”

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Curvature instability - S. Leibler (‘86) membrane inclusions induce a “spontaneous curvature” Near the inclusion One refinement for “biological” membranes Equilibrium distribution of inclusions follows the curvature Effective bending rigidity If the rigidity is <0 the membrane spontaneously curves Unstable for any inclusion density

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Curvature instability - formalism - membrane inclusions induce a “spontaneous curvature” Near the inclusion Inclusion density Equilibrium distribution of inclusions follows the curvature Effective bending rigidity If the rigidity is <0 the membrane spontaneously curves ~ Landau expansion Effective attraction

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Physical interactions between membrane inclusions Attraction between junction mediated by the membrane deformation Bruinsma etal. ‘94 Attraction / Repulsion - Goulian etal. ‘93 Effect on Thermal Fluctuations (Casimir force) Attraction - Goulian etal. ‘93 - Kardar etal. ‘98 Asymmetric inclusions Cell Junction Many-body interactions Oster etal. ‘99

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Quasi-spherical “Soft” Shells Thermal Self-Assembly of Caveolin aggregates Internal Structure - Striated Coat Asymmetrical Interaction between Aggregates Physical Origin ? Structures of Caveolae cell membrane cell interior clathrin-coated vesicle caveolae Location Plasma membrane of many cells: Endothelial cells, adypocytes, cardiac muscles… Function Many: Endocytosis - ligand binding Interaction with signaling proteins - cholesterol transport…

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Caveolae pictures 100 nm. T. Fujimoto etal. J. Electron Microscopy 47, 452 (1998) Electron micrograph of caveolae in the rat smooth muscle cell Barr=100nm As C M . Gumbleton Adv. Drug Delivery Rev. 49 (2001) 281 TEM of the alveolar-pulmonary capillary barrier in rat lung (As: Alveolar space; C: capillary lumen)

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Hypothetical model of the principal actions of caveolae and caveolins in signaling. Left of dashed line: The major part of caveolins (brown) is present as oligomers in structurally defined caveolae. Filamin (turquoise)– caveolin interactions link some caveolae to actin filaments (tan). Caveolin molecules with a ligand-binding site (scaffolding domain) not involved in oligomer formation can instead sequester and inhibit signaling proteins such as H-Ras (yellow). Activated growth factor receptors (blue-gray) in caveolae recruit adaptor proteins (red-white) like Grb2 and mSOS and can activate caveola-resident H-Ras. Outside of caveolae a fraction of caveolin-1 associates with integrins (gold) and keeps Src-family kinases like Fyn (orange) in an inactive conformation. Upon cell–matrix adhesion (integrin ligation) caveolin-1 and Fyn are coclustered with the integrins, and in the presence of GPI-linked uPAR (red) glycolipid rafts are recruited to the adhesion site. Fyn is activated and the inhibitory action of caveolin-1 is relieved. Fyn signals, via adapter molecules (Shc, Grb2/mSOS), to H-Ras. The activation of H-Ras (in rafts or caveolae) eventually leads to the activation of MAP kinases and signaling to the cell nucleus. K-Ras (light yellow) is present in a different membrane compartment from caveolins due to a polybasic region (pink) and takes part in different signaling events. Raft and caveola membranes are indicated in green. Small vertical lines within the noncaveolar membranes indicate cholesterol concentration. Fyn, mSOS, and Ras associate with the plasma membrane via lipid modifications (black dots). Right of dashed line: Cholesterol depletion of the membrane leads to the loss of the caveolin coat from the membrane. Concomitantly caveolae and functional rafts disappear. This prohibits the local enrichment of H-Ras, Src kinases, adapters, and uPAR. Thus, signaling via caveolin is abolished. Experimental Cell Research 261, 111–118 (2000) Martin Stahlhut, Kirsten Sandvig and Bo van Deurs

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CAVEOLAE - Membrane invagination // Main constituant: protein Caveolin Protein Structure Peripheral membrane protein C-terminal 135-178 a.a N-terminal 1-101 a.a transmembrane domain 102-134 attractive part 61-101 a.a membrane Homo-oligomer ≈ 14-16 caveolin Caveolin protein Caveolin - 178 aa Caveolin aggregates 14-16 molecules : 4-6 nm Caveolae Bud 50-80 nm from Schlegel - Lisanti Cell Signal 10, 457 (1998)

STRATEGY : 

STRATEGY Model for Caveolin - force distribution Membrane deformation - Interactions between proteins Protein organization I : Oligomer formation Protein organization II : Bud formation Protein organization III : Stripes of proteins Comparison with experimental observations

MODEL FOR CAVEOLIN OLIGOMERSForce on the membrane : 

MODEL FOR CAVEOLIN OLIGOMERSForce on the membrane Polymer chain grafted on a wall r monomer concentration (Thermal) Fluctuations force (pressure) on the wall Force distribution for the polymer entropic effect (thermal fluctuations of the chain) Caveolin protein on membrane Applied force for the protein complex ???

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and we know the lengthscales Not possible to calculate the force distribution No net force / no net torque Origin for pressure in caveolin brushlets: Thermal fluctuation (small if rigid) Steric constraint - Bad solvent effect Most of the force is in the center we know the symmetries of the force We can estimate the strength of the force

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PROTEIN-INDUCED MEMBRANE DEFORMATION-MEMBRANE-MEDIATED PROTEIN INTERACTIONS Important physical parameters Surface tension Bending Rigidity

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Membrane-mediated Interactions (exact) Small repulsive interaction Input : membrane deformation energy Output : membrane deformation Interactions between inclusions

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PROTEIN ORGANIZATIONOligomer formation - Bud formation - Stripes of proteins

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Thermal aggregation - cf. Micelle formation in surfactant solution Input: energy in aggregate fp Output : Aggregate size p* and critical aggregation concentration (c-a-c) Densities p p 5nm 100nm

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Oligomerisation ~ 2-D micellisation process But, theory is questionable for small aggregates details of the protein attraction may matter Sticking energy polymer brush entropic repulsion - a=3/2 Energy gain per protein Proteins on the outskirts don’t get full contact Oligomer formation N-terminal attractions Q proteins per oligomer Energy per protein has a minimum crowding To obtain experimental values we need Reasonable numbers Repulsion: entropic energy scale Attraction: Hydrogen bond energy scale

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Bud energy per brushlet Bud formation - In-plane phase separation Driving force: preferred curvature * cac R

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Protein concentration for budding (very) sensitive to surface tension Role in cell mechanosensitivity Equilibrium radius variation with surface tension equilibrium bud radius E0=10kBT concentrations There is an optimal bud size (≠ curvature instability) Size ~ insensitive to membrane tension Physiological g Results

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Striped distribution of protein oligomers Origin for striated coat ? Interactions between oligomers Short Range specific attraction mediated by distal third region of C-terminal (10 aa) b~ few nm k-1~30 nm Erep~10-2 kBT Eattp~3-4 kBT Long range physical repulsion between inclusions and

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Perturbation in Fourier space Optimal size A few brushlet dimension Liquid-gas transition Strength of the attraction - few kBT entropy interaction Microphase separation Strength of the repulsion - 10-2 kBT enough !

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CONCLUSION Three levels of organisation : Caveolin aggregation into brushlets Concentration of brushlets in membrane pits Striations at the surface of the pits can be explained by simple physical arguments Comparison with mutational analysis of caveolin-induced vesicle formation Shengwen Li1 etal. FEBS Letters 434 (1998) 127 Deletion of sticky part on N-terminal No oligomer (brushlets) strong reduction of typical energy scale E0 Deletion of main part of C-terminal No (weak) short range attraction between oligomer increase of interaction parameter (virial coef) Both mutans are able to drive vesicle formation, but much larger vesicles ~ 1µm Qualitatively consistent with our results Testable predictions Effect of membrane tension... cf. Role in cell mechanosensitivity